The stratified squamous epithelium of the oral cavity is self-renewing and has a resident keratinocyte stem cell population necessary for generating new cells to replenish the terminally differentiated cells lost from the oral surface. These stem cells and their derivative progenitors are thought to be scattered along the inner most basal layer, where they lie dormant in a characteristic slow-cycling state. The inherent ability of the stem cells to retain labeled DNA for a prolonged time period makes it possible not only to mark their location within the basal layer and visualize them, but also to prospectively purify them. In a strategy analogous to a BrdU pulse- chase experiment, a regulatable Histone2B-GFP transgenic mouse model has been utilized to fluorescently label quiescent stem cells in vivo. As a complementary approach, specific markers, if available can also be utilized to identify stem cells. However, the lack of ideal surface markers has been an impediment for the isolation and subsequent biological and biochemical characterization of epithelial stem cells, particularly in the oral cavity. Recent studies have offered compelling evidence in support of the transcription factor p63, particularly the ?Np63 isoform, to be highly expressed in the stem cell compartment of the stratified squamous epithelium. In addition, extensive analysis of p63-deficient mice has revealed the essential role of this factor in the self-renewal of keratinocytes. We have generated a novel ?Np63-GFP reporter mouse model system that allows to mark the cells that express high levels of ?Np63, which we hypothesize are likely to represent stem cells. The availability of the Histone2B-GFP and ?Np63-GFP animals will allow us to identify and sort the prospective stem cells from the mouse oral epithelium at discrete anatomical sites such as buccal mucosa, palate and gingiva. Importantly, our strategy will permit the isolation of viable stem cells that can be subsequently analyzed in clonogenicity and molecular profiling studies. Finally, we hope to identify additional and long-sought markers of oral stem cells, which will undoubtedly be of crucial importance for proper clinical application of oral epithelial cultures in cell therapy as well as for studies on epithelial cancer stem cells and tumorigenesis. More importantly, the data obtained from our murine studies will be invaluable for planning and executing similar stem cell studies using human samples. PUBLIC HEALTH RELEVANCE: Oral stratified epithelia continuously generate new cells from stem cells (SCs) to replenish the differentiated cells that are sloughed off from the surface. Unlike other organs and tissues, our knowledge about SC biology and function in the oral mucosa is limited - therefore, there is a growing need to identify, isolate, and characterize SCs of the oral epithelia. By using two complementary genetic strategies, we plan to isolate the oral SCs, test their distinctive self- renewing properties and generate their distinctive molecular fingerprints to better understand the biology of oral stratified epithelia and harness the regenerative power of SCs.